(1) Goals of Project: - To identify HIV-1 subunits which will generate widely cross neutralizing antibodies. - To identify a carrier which will increase the immunogenicity of the HIV-1 subunits, and will be able to recall anti-HIV B memory cells in patients with pre- existing immune deficiency. - To identify a carrier which would augment the TH1/TH2 ratio of infected individuals and will favor generation of cellular responses including cytotoxic cells (CTL). (2) Experimental approaches: - The gram negative Brucella abortus (Ba), and LPS derived from its cell wall (Ba- LPS), were tested as carriers for either inactivated HIV-1 virions, gp120 (SF2) glycoprotein, or peptide derived from the V3-loop of HIV-1 (MN) env. The different conjugates were used to immunize mice with different degrees of T cell deficiency. -In vitro studies with PBL from normal human as well as HIV-1 infected patients were assessed for their lymphokine production in response to Ba and Ba-LPS. Both PCR and biological assays were used. (3) Major findings: - Ba conjugated to a peptide containing B-cell epitope and CTL epitope (N3v3), generated both neutralizing antibodies and cytotoxic T cells capable of killing HIV-infected targets. CD4- depleted mice retained their ability to generate anti-HIV neutralizing Ab and CTL after immunization with the Ba-N3V3 conjugate. - From a safety point of view, Ba or its LPS are much less toxic to animals than E. Coli derived LPS. - Ba and Ba-LPS were found to directly activate purified human CD4-positive TH1 cells, and to a lesser degree, CD8-positive cells, as judged by induction of the lymphokines IL2 and IFN-gamma. PBL from HIV-1 infected individuals were also responsive. Ba and Ba-LPS can also activate IL12 secretion by human monocytes. Publications: (1) B. Golding, J. Inman, P. Highet,, R. Blackburn, J. Manischewitz and H. Golding 1955. J. Virology 69:3299-3307. (2) M. Zaitseva, H. Golding, M. Brtts, A. Yamauchi, E. Bloom, L. Butler, L. Stevan, and B. Golding. 1995 Infec. and Immunity. 63: 2720-2728